Current recommendations for the molecular evaluation of newly diagnosed holoprosencephaly
patients.

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American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 154C:93 – 101 (2010)
A R T I C L E
Current Recommendations for the Molecular
Evaluation of Newly Diagnosed
Holoprosencephaly Patients
DANIEL E. PINEDA-ALVAREZ, CHRISTÈLE DUBOURG, VÉRONIQUE DAVID, ERICH ROESSLER,
AND MAXIMILIAN MUENKE*
Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain in humans and
is typically characterized by different degrees of hemispheric separation that are often accompanied by similarly
variable degrees of craniofacial and midline anomalies. HPE is a classic example of a complex genetic trait with
‘‘pseudo’’-autosomal dominant transmission showing incomplete penetrance and variable expressivity. Clinical
suspicion of HPE is typically based upon compatible craniofacial findings, the presence of developmental delay or
seizures, or specific endocrinological abnormalities, and is then followed up by confirmation with brain imaging.
Once a clinical diagnosis is made, a thorough genetic evaluation is necessary. This usually includes analysis of
chromosomes by high-resolution karyotyping, clinical assessment to rule-out well recognized syndromes that are
associated with HPE (e.g., Pallister-Hall syndrome, Smith-Lemli-Opitz syndrome and others), and molecular
studies of the most common HPE associated genes (e.g., SHH, ZIC2 and SIX3). In this review, we provide current
step-by-step recommendations that are medically indicated for the genetic evaluation of patients with newly
diagnosed HPE. Moreover, we provide a brief review of several available methods used in molecular diagnostics of
HPE and describe the advantages and limitations of both currently available and future tests as they relate to high
throughput screening, cost, and the results that they may provide. Published 2010 Wiley-Liss, Inc.{
KEY WORDS: holoprosencephaly; HPE; disease genes; multi-factorial inheritance; molecular diagnostics
How to cite this article: Pineda-Alvarez DE, Dubourg C, David V, Roessler E, Muenke M. 2010.
Current recommendations for the molecular evaluation of newly diagnosed holoprosencephaly patients.
Am J Med Genet Part C Semin Med Genet 154C:93–101.
INTRODUCTION
Holoprosencephaly (HPE) is the most
common disorder of the developing
forebrain in humans, occurring with a
frequency of 1:250 conceptuses [Matsunaga and Shiota, 1977] and 1:10-16 000
live births [Roach et al., 1975]. The
HPE phenotypic spectrum results from
failure of the forebrain to cleave into two
Dr. Pineda-Alvarez is a Colombian trained medical graduate who is currently a Clinical
Molecular Genetics fellow in the Medical Genetics Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda, MD, USA.
Dr. Dubourg is a faculty member in the branch ‘‘Génétique des pathologies Liées au
Développement’’, UMR 6061 CNRS, IGDR, University of Rennes 1, Faculty of Medicine, Rennes,
France, and a hospital member in the Laboratory of Molecular Genetics, CHU Pontchaillou,
Rennes, France. Her diagnostic and research interests include holoprosencephaly (HPE) and
mental retardation.
Professor David is the chief of Holoprosencephaly group in the Branch of ‘‘genetics of
developmental pathologies’’, UMR 6061 CNRS IGDR, Faculty of Medicine, University of Rennes 1,
France. She is the chief of the Molecular Diagnosis laboratory, CHU Pontchaillou, Rennes, France.
Her research interest mainly concerns HPE, and her clinical research also focuses on iron overload
genetic diseases.
Dr. Roessler is a faculty member in the Medical Genetics Branch, National Human Genome
Research Institute, National Institutes of Health, Bethesda, MD, USA. His research interests
include malformations of the forebrain associated with HPE as well as disturbances in organ
sidedness, or laterality.
Dr. Muenke is the Branch Chief of the Medical Genetics Branch. His research interests include
HPE, craniofacial malformation syndromes, and Attention Deficit Hyperactivity Disorder (ADHD).
*Correspondence to: Maximilian Muenke, Medical Genetics Branch, National Human Genome
Research Institute, National Institutes of Health, 35 Convent Drive, MSC 3717, Building 35, Room
1B-203, Bethesda, MD 20892-3717. E-mail: mamuenke@mail.nih.gov
DOI 10.1002/ajmg.c.30253
Published online 26 January 2010 in Wiley InterScience (www.interscience.wiley.com)
Published 2010 Wiley-Liss, Inc.
{
This article is a US Government work and, as such, is in
the public domain in the United States of America.
hemispheres. Different degrees of hemispheric separation, ranging from the
classically described alobar form, to
semilobar, lobar and middle-interhemispheric variant (MIHV) describe the
anatomically distinguishable forms of
HPE. The mildest end of the spectrum
includes subtle midline brain anomalies.
These phenotypes are often accompanied by a broad spectrum of craniofacial
differences, ranging from the most
severe form with cyclopia (one eye) or
synophthalmia (two fused eyes) with a
proboscis (nose-like appendage), to less
severe forms with hypotelorism, midface hypoplasia or a single maxillary
central incisor (SCI) [Muenke and
Beachy, 2000; Cohen, 2006; Dubourg
et al., 2007; reviewed in Solomon et al.,
2010]. The occurrence and manifestations of HPE are influenced by both
genetic causes and environmental risk
factors. In cases where a specific gene is
known to be causative, it is inherited
as a complex trait with incomplete
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AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
penetrance and variable expressivity.
The basis of these phenotypic differences
is largely unknown but likely reflects
measured and unmeasured genetic and
environmental components [Solomon
et al., 2009].
CYTOGENETIC
ALTERATIONS AND
MUTATIONS OF
DEVELOPMENTAL GENES
ARE THE MOST COMMON
KNOWN CAUSES OF HPE
It is estimated that the cause of HPE is
due to cytogenetic anomalies in 30–50%
of individuals, to well recognized syndromes (e.g., Smith-Lemli-Opitz syndrome (SLOS)) in 25%, to either
environmental causes and/or unknown
genetic alterations in 10–15%; and to
mutations in established HPE gene(s) in
5–10% [Bullen et al., 2001; Dubourg
It is estimated that the cause
of HPE is due to cytogenetic
anomalies in 30–50%
of individuals, to well
recognized syndromes (e.g.,
Smith-Lemli-Opitz syndrome
(SLOS)) in 25%, to either
environmental causes and/or
unknown genetic alterations in
10–15%; and to mutations
in established HPE
gene(s) in 5–10%
et al., 2007; Ong et al., 2007; Roessler
et al., 2009a]. Additional risk factors that
may act alone or in concert with genetic
alterations include the use of retinoids,
statins, or alcohol during pregnancy,
alterations in the biosynthesis of cholesterol, and pre-existing or gestational
diabetes [Cohen and Shiota, 2002].
Mutations in at least 12 genes have
been detected in patients with HPE;
however, there is significant variability
in the observed mutation rate of each
gene (see below). The most common
HPE genes were identified as mutational
targets within loci defined by chromosomal rearrangements [Muenke and
Beachy, 2000; Dubourg et al., 2004].
Among the best characterized HPE
genes are SHH [Roessler et al., 1996],
ZIC2 [Brown et al., 1998], SIX3 [Wallis
et al., 1999], TGIF [Gripp et al.,
2000], GLI2 [Roessler et al., 2003],
PATCHED-1 [Ming et al., 2002],
DISP1 [Roessler et al., 2009c]. Most
CLIA-certified laboratories, both commercial fee-for service and those associated with National Institutes of Health
(NIH) or similar centers, only screen the
first four genes (the named HPE loci
2–5) for mutations on a routine basis.
Microdeletions and microduplications
have been suggested to play important
roles given that some of these alterations
occur in the vicinity of known HPE
genes [Bendavid et al., 2009].
Currently, there is still a large
proportion of individuals with nonsyndromic and non-chromosomal HPE
(20–10% of all HPE patients) in
whom no specific genetic cause can be
identified [Wallis and Muenke, 2000].
The general consensus regarding the
etiology of HPE is that the molecular
interactions and pathways are complex
[Monuki, 2007], consistent with the
theory that a large number of loci or
genetic factors are yet to be identified
and fully understood. The primary goal
of this review is to describe the current
recommendations for molecular genetics testing of patients with newly diagnosed HPE, the types of strategies for
evaluation that are currently used, what
tests are likely to be of use in the future,
the advantages and limitations of these
technologies, and the importance and
benefits of the participation of the
patients and their families in research
studies.
CURRENT EVALUATION
STRATEGY
The clinical diagnosis of HPE is confirmed by a combination of physical
examination, family history, and brain
imaging (MRI, CT, or ultrasound, etc.).
Once HPE is clinically confirmed,
ARTICLE
parental samples should also be obtained
in order to allow for a better interpretation of results in the setting of a positive
cytogenetic or molecular finding in the
proband.
As shown in Figure 1, we propose a
general strategy for the genetic evaluation of a patient newly diagnosed with
HPE. HPE is usually diagnosed clinically
based upon specific phenotypic features
(described above) [Cohen, 2006;
Dubourg et al., 2007; Orioli and
Castilla, 2007], which typically must
then be confirmed with brain imaging in
order to fully characterize the anomaly
[Hahn and Plawner, 2004]. A comprehensive evaluation of a patient with HPE
should typically begin with cytogenetic
studies, including a high-resolution
karyotype with a minimum of 550 band
resolution, given that 30–50% of
patients will have a chromosomal anomaly, including deletions, duplications,
but also balanced translocations and
inversions that are not detected by array
comparative genomic hybridization
(array CGH) [Bullen et al., 2001;
Cohen, 2006; Ong et al., 2007; Orioli
and Castilla, 2007]. In selected patients,
medically indicated studies should then
be done to rule out syndromes that
might cause HPE (e.g., elevated 7dehydro-cholesterol levels in SLOS).
Finally, in all nonsyndromic patients
found to have normal chromosomes,
molecular analysis should be performed
for the most common genes implicated
in HPE: SHH, ZIC2 and SIX3 [Wallis
in all nonsyndromic patients
found to have normal
chromosomes, molecular
analysis should be performed
for the most common genes
implicated in HPE: SHH,
ZIC2, and SIX3
and Muenke, 2000; Dubourg et al.,
2004].
Parental samples should be obtained
at the initial evaluation of a proband as
the study of these individuals can be
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AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
95
Figure 1. Algorithm for the genetic study of new holoprosencephaly patients. Bold lines refer to medically indicated tests; thin lines are
optional tests depending on a specific clinical indication or the capabilities of the diagnostic laboratory; dotted lines refer to tests available in
research labs that will contribute to a better understanding of HPE. For further details, see the following references: a: [Hahn and Plawner,
2004], b: [Dubourg et al., 2007], c: [Roessler and Muenke, this issue], d: [Bendavid et al., this issue], e: Refers to High-Resolution DNA
Melting (HRM), f: Multiplex Ligation-dependent Probe Amplification (MLPA), g: gene specific phenotype, h: [Bullen et al., 2001; Ong
et al., 2007], i: recruit parental samples for better test interpretation in case of a positive result. þ: positive diagnostic test results.
critical for the interpretation of the
proband’s test results and for future
genetic counseling, whether or not a
cytogenetic or molecular diagnosis is
immediately established. The strongest
predictors of the pathogenicity of new
alterations relates to whether the changes
are de novo gross cytogenetic, microdeletions/duplications, or mutations
[reviewed in Roessler and Muenke,
2010].
For newly diagnosed patients who
have an abnormal karyotype, the cytogenetic findings should be correlated
with the clinical phenotype and the
underlying mechanism involved. For
example, well recognized trisomies
involving chromosomes 13 and 18 or
rearrangements that disrupt one of the
major genes implicated in HPE, such as
SHH or ZIC2, would be expected to
contribute to the etiology of HPE
[Dubourg et al., 2007]. Other chromo-
somal rearrangements can also occur [see
Roessler and Muenke, 2010]; however currently there is little proof of the
pathogenicity for the majority of them.
New technologies, controlled population genetics, and functional studies
should allow us to further expand our
knowledge. Again, parental studies are
important to define whether the anomaly is segregating through the family or if
it is a de novo event. In the case of
chromosomal rearrangement, more indepth molecular analysis of the chromosomal breakpoints, using DNA sequencing or array-CGH, can be important
given that the vicinity of the breakpoints
produces unstable DNA with deletions
and duplications frequently occurring
beyond the particular locus. These additional studies will allow better characterization of the genetic alterations and
phenotypic correlations, which may be
helpful for genetic counseling purposes.
For the patients with a normal
karyotype, DNA sequencing analysis
should be performed for the most
commonly identified genes associated
with HPE. In general, mutations in
SHH are present in 12% of these
patients [Roessler et al., 2009a], ZIC2
in 9% [Roessler et al., 2009b], and
SIX3 in 5% [Lacbawan et al., 2009].
Given the high detection rate of likely
pathologic mutations, we consider these
genes to be essential for a first line
medical assessment. Other genes have
been described to be implicated in HPE,
such as TGIF (altered in 1% of
patients) [Gripp et al., 2000; Wallis and
Muenke, 2000], GLI2 (1%) [Roessler
et al., 2003], PATCHED-1 [Ming et al.,
2002], DISP1 [Roessler et al., 2009c],
FOXH1, NODAL [Roessler et al.,
2009d], and others. However, at the
present time, we recommend that
these latter genes with low mutation
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AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
frequency rates among HPE patients be
tested only in selected cases, or that they
be referred to specialized testing centers
with the requisite expertise.
One example of a specialized situation that calls for testing of GLI2 is
when specific abnormalities occur in the
development of the pituitary gland, in
the context of variable brain and craniofacial anomalies consistent with the
broad spectrum of HPE [Roessler et al.,
One example of a specialized
situation that calls for testing
of GLI2 is when specific
abnormalities occur in the
development of the pituitary
gland, in the context of variable
brain and craniofacial
anomalies consistent with the
broad spectrum of HPE
2003; Roessler et al., 2005]. Likewise,
other genes have also been shown to be
associated with characteristic brain and
craniofacial abnormalities [Solomon
et al., 2010; Muenke Lab, unpublished
data]. In these special cases where a
specific phenotype is present, molecular
analysis of the associated locus is considered medically indicated.
From the current molecular diagnostic perspective, exonic mutational
analysis via bi-directional DNA
sequencing remains the gold standard.
Both fee-for-service and free-of-charge
CLIA-certified testing based in research
laboratories (e.g., at the NIH in the
United States) are available and give
comparable results, which can be used
for the clinical management of patients
and for genetic counseling for the family.
When a novel mutation is identified in a
proband, such as a single nucleotide
change, insertion, duplication, deletion
or a frame-shift mutation, parental
samples should subsequently be tested
to assess whether the mutation is segregating in the family (familial HPE)
[Solomon et al., 2009] or a de novo
variant. In general, de novo mutations are
more likely to be pathogenic based on
functional studies [Domene et al., 2008].
However, a large proportion of patients
have unique mutations that are familyspecific which can make it very difficult
to predict the functional consequences
[Roessler et al., 2009a]. Parental studies
should also be conducted even if the
identified mutation has previously been
associated with HPE in order to provide
the family with accurate recurrence risk
information.
In order to better identify which
genetic variants are truly pathogenic, the
identified variants must be correlated
with their predicted or experimentally
determined residual function. Computerized prediction algorithms may be
used; however these algorithms may
be inconclusive, and therefore, highly
specialized functional studies based on
animal models, cellular models and
conservation analyses among vertebrate
species are typically required. Functional
consequences of changes in SIX3
[Domene et al., 2008], SHH [Roessler
et al., 2009a], ZIC2 [Roessler et al.,
2009b], and TGIF [El-Jaick et al., 2007],
have been well illustrated [reviewed in
Roessler and Muenke, 2010].
Not all variants among the HPE
genes are obvious loss-of-function.
Although nucleotide changes occurring
in very conserved regions of the genome
are more likely to cause defects through
loss-of-function, further analyses are
frequently necessary to determine their
precise effects [Kryukov et al., 2007].
Importantly, there is also increasing
evidence that gene regulatory elements
and non-coding portions of HPE genes
can play an important role in disease
Importantly, there is also
increasing evidence that gene
regulatory elements and
non-coding portions of HPE
genes can play an important role
in disease causation and would
be missed by most traditional
diagnostic strategies
ARTICLE
causation and would be missed by most
traditional diagnostic strategies [Jeong
et al., 2008].
Local genetic counseling, facilitated
by the expertise of tertiary care centers
(such as The Carter Centers for Brain
Research in HPE and Related Malformations, including the NIH) and patient
groups (‘‘Families for HoPE,’’), should
be offered to families whether results of
the genetic tests are negative or positive.
This counseling should be performed
based upon state-of-the-art evidence to
help to interpret the results and their
limitations. When there is inconclusive
evidence about the effect of a given
variant, it should be made clear to the
family that the effect is uncertain.
Finally, we recommend clinicians to
assess the willingness of the parents to
participate in research in HPE, which is
beneficial to the final and broad goal of
larger knowledge and better medical
management for their children. Moreover, research gives the opportunity for
patients to be evaluated by an expert
multidisciplinary team, which can advise
parents and local physicians in the
appropriate treatment of underlying
disorders.
PAST, PRESENT, AND
FUTURE METHODS AND
THEIR IMPLICATIONS
In Table I, we present the advantages and
disadvantages of several methods that
have been used and that are being
proposed for the molecular study of
HPE. In the past, SSCP and DHPLC
have been used as effective screening
methods [Roessler et al., 1996; Brown
et al., 1998; Wallis et al., 1999; Gripp
et al., 2000; Dubourg et al., 2004]. SSCP
was initially the best way to pre-screen
individuals for variants for a given DNA
product, however it was not an ideal
technique given that some materials
required special handling and training
and constituted a potential hazard for the
laboratory environment, and that the
sensitivity of the test was low [Orita
et al., 1989]. DHLPC was presented as
an alternative method, as it had
improved sensitivity, provided higher
throughput options than SSCP, and
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AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
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TABLE I. Past Present and Future Molecular Diagnostic Methods
Use
Past
Method
Advantages
Disadvantages
A popular, rapid, inexpensive screen for
nucleotide variants
Detects presence of normal and variant
alleles
Heterozygous vs. homozygous results
obvious
Semi-automated
Higher throughput capacity and
sensitivity than SSCP
Requires less time and labor than SSCP
Improved cost profile
Lowest sensitivity and specificity
Small amplicons
Use of acrylamide gels and radioactivity
Requires confirmation with sequencing
Automated capillary DNA
sequencing
Gold Standard
100% sensitivity and specificity
Semi-automated
High throughput capacity
HRM
(High-resolution DNA melting)
Effective screening method
High throughput
Sensitivity over 90%
Specificity excellent and improves with
increased throughput
Post-PCR manipulation is not required
Fast automatic run where the analysis
can focus on sequencing the
uncommon variants flagged by the
software
Fast and high throughput method
Detects sub-microscopic deletions/
duplications missed by sequencing
Capable of detection of genome wide
gains/losses of copy
Requires no hypothesis
Requires significant investigator edits
Ambiguities frequent, occult allelic
drop
Typically fails to detect large deletions
or duplications
Screening of some GC-rich regions can
be challenging
Optimal results with small amplicons
300 bp
Requires follow-up sequencing of
variants
SSCP
(Single strand conformational
polymorphism)
DHPLC
(Denaturing high-performance liquid
Chromatography)
Present
MLPA (multiplex ligation dependent
probe amplification)
aCGH
(Array comparative genomic hybridization)
Future
Next-Generation Sequencing
the preparation, run and analysis of
the experiments were relatively short
[O’Donovan et al., 1998]. Nevertheless,
due to its good sensitivity, DHLPC is still
used in many diagnostic laboratories
around the world.
Capillary electrophoresis DNA
sequencing is the current gold standard
for mutational screening of HPE genes,
with its primary advantage being close to
Capable of genome-wide
individualized data
High tiling path ¼ few errors
Unambiguous results
Relatively fast
100% sensitivity and specificity. However, data analysis is labor intensive and
its interpretation may be challenging due
to presence of variants of unknown
significance. Furthermore, allelic dropout and failure to detect deletions/
duplications that are larger than the
sequence being interrogated may occur.
Although DNA sequencing is more
readily available than other technologies,
Specificity still marginal
Requires confirmation with
sequencing
Typically requires validation studies
Few laboratories perform test
Expensive
Validation with another method often
needed
Only large scale changes
Needs several micrograms of DNA
In development on multiple platforms
Huge amounts of data (almost all of
which is normal)
Significance of most variants will
initially not be understood
and it can be used with equal success on
both medically indicated and researchonly genetic tests, there is still a strong
need for newer methods given the
extensive heterogeneity of causative
HPE genes.
Multiplex
ligation-dependent
probe amplification (MLPA) (MRCHolland, Amsterdam) is a relatively
new molecular method to detect the
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AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
occurrence of micro deletions/duplications in genes. There is a panel commercially available (SALSA MLPA
kit P187 Holoprosencephaly—MCRHolland, Amsterdam, Netherlands)
with probes spanning the 8 HPE genes
[Bendavid et al., 2009]. Among the
limitations of this method are that it is
available in only a few laboratories, a
follow-up test is necessary to validate
presence of dosage differences (e.g.,
qPCR and Fluoresce In Situ Hybridization—FISH), and it is unable to
detect single nucleotide mutations or
smaller deletions or duplications. There
is sufficient evidence in the literature of
an overwhelming number of single
nucleotide mutations or small deletions/duplications causing truncated
proteins. For example, in a recent study
on patients with HPE and alterations in
SHH, there were 125 different mutations tabulated, none of which are
detected by MLPA [Roessler et al.,
2009a]. Hence, copy number variations
and hypothetical promoter or enhancer
variations are likely to be among the least
common types of variations that are
likely to be detected.
High-resolution DNA melting
(HRM) strategies have recently been
proposed to pre-screen samples for
mutations [Reed et al., 2007]. In our
experience, amplicons from many individuals can be simultaneously screened
from genomic DNA in roughly 2 hr,
followed by direct sequencing of a
targeted subset of presumed variants.
This method promises considerable savings in terms of money and time in the
identification of variants. Some of its
greatest advantages are the high sensitivity and specificity (over 95% for
heterozygous
variants)
[Wittwer,
2009], as well as its high throughput
nature (up to 384 samples to be screened
per run, in a Roche LightCycler 480 II
instrument (Roche Appplied Science,
Indianapolis, IN and Idaho Technology
Inc., Salt Lake City, UT)). HRM loses
efficacy when screening GC-rich amplicons due to the difficulty in denaturing
them. However, denaturing solutions,
such as Roche GC-RICH solution
(Roche applied science, Indianapolis,
IN), can be added to enhance the
melting process to increase the specificity [Tindall et al., 2009]. Moreover,
current protocols recommend that
amplicons be limited in size, up to 400
base pairs [Wittwer, 2009].
Next-Generation (NextGen) sequencing strategies and array-CGH
(aCGH) offer the promise of great
amounts of information [Bejjani and
Shaffer, 2006; Mardis, 2008] although it
is not yet clear which of the many new
methods will emerge as the most useful.
Next-Generation (NextGen)
sequencing strategies and
array-CGH (aCGH) offer the
promise of great amounts of
information although it is not
yet clear which of the many
new methods will emerge
as the most useful.
With both of these new strategies, the
interpretation of results should be made
carefully, since miscalling of normal
variants as mutations presents the risk
of misinterpretation. Currently, there is
not enough evidence from well-controlled studies to unambiguously differentiate disease-causing alterations from
incidental copy number variants
(CNVs) except for the ones involved in
the known HPE genes [Bendavid et al.,
2009]. Further research should help to
mitigate these obstacles. Since these
techniques are so new and rapidly
changing, most of the technologies have
not been FDA approved for routine
clinical use, but are nevertheless currently used by commercial diagnostic
laboratories, such as GeneDx (Gaithersburg, MD, USA) for the diagnostic
studies of several diseases. Array-CGH
may not be appropriate for use on a
routine basis until there is a better
understanding of the implications of
CNVs in the pathogenesis of HPE. As
with all detection methods, presumptive
positive results should be followed up by
family studies, since occurrence of novel
events are more likely to be pathogenic.
ARTICLE
New technologies, such as HRM,
aCGH and NextGen Sequencing, will
allow for the generation of large
amounts of data with sensitivities and
specificities over 90%, the ability to
detect CNVs that were not previously
identifiable, and for the routine screening of more genes and regulatory
elements to be both cheaper and faster.
However, the generation of such overwhelming amounts of data by itself does
not always translate into a better understanding of a disorder. Consequently, the
application of these tests in a clinical
context is presently limited [Bejjani and
Shaffer, 2006].
BENEFITS OF RESEARCH IN
PATIENTS WITH
HOLOPROSENCEPHALY
The current knowledge of HPE is the
product of nearly four decades of
research in several specialized centers.
Despite that, our knowledge is still very
incomplete, and many important questions remain. To better understand this
complex disorder, patients and their
families should continue to be encouraged to freely enroll in these studies,
whether they have positive or negative
genetic testing results.
To better understand this
complex disorder, patients and
their families should continue
to be encouraged to freely enroll
in these studies, whether
they have positive or negative
genetic testing results.
The participation of a diverse set of
parent–child trios, extended families
and well-controlled case-control studies
will allow for future work to address new
genetic associations, modifier screens,
and other methods aimed at better
understanding how genetic interactions,
genetic variations, and environmental
co-factors may influence the variable
penetrance and expressivity of HPE
traits, even when a mutation is present
ARTICLE
in a well-characterized gene. Hence, the
participation of parents in the molecular
evaluation of their children can have
both direct and indirect benefits for HPE
research.
Additionally, there are a large number of genomic variants in which the
biological effects are unclear. Advances
in technology and continued cooperation with research centers can often
result in the development of functional
tests for novel sequence variants in
known and newly identified HPE genes
in order to clarify the nature of such
alterations and their implications in a
clinical context.
Our experiences over the past
decade have proven that the value of
cooperation amongst multiple international testing centers goes beyond the
simple ability to share methodologies
and testing strategies, but that the
sharing of patient data and test samples
enhances the likelihood of identifying
additional HPE genes and the understanding of their associated phenotypic
manifestations.
One of the benefits of enrolling in a
research study is that as new genes are
identified, patients in whom mutations
were not previously identified can be
tested for the new genes. If families
consent, they are notified of these novel
results, giving them the opportunity to
be counseled based on new state-of-theart evidence.
In the long-term, research will
allow a more integral understanding of
HPE, in which children and parents will
directly benefit through better counseling and individualized clinical management, focusing on specific issues arising
from a genetic variant and its interactions.
CALL FOR A
HOLOPROSENCEPHALY
CONSORTIUM
We recommend the formation of a
worldwide consortium where research
data, DNA samples and cell lines would
be shared between the largest possible
number of active investigators involved
on HPE research in order to accomplish
an integration of knowledge that would
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
contribute to a thorough understanding
of the clinical and genetic aspects of this
disease. While no such formal organization yet exists, the rationale for such
an effort is clear. The extensive genetic
We recommend the formation
of a worldwide consortium
where research data, DNA
samples and cell lines would be
shared between the largest
possible number of active
investigators involved on HPE
research in order to accomplish
an integration of knowledge
that would contribute to a
thorough understanding of the
clinical and genetic aspects of
this disease. While no such
formal organization yet exists,
the rationale for such
an effort is clear.
heterogeneity of HPE and the unresolved issues underlying its characteristic
variable expressivity compel researchers
in this field to cooperate with one
another and to enlist the cooperation of
primary care providers, patient groups,
and families in this effort. Some of the
obvious future challenges of this proposed group will be to collect cases for
large-scale studies (e.g., to establish
routine functional studies based on
animal or cellular models, perform
family-based association studies, and
case-control association studies) to dissect the genomic variants that impact on
HPE incidence and severity. Large datasets increase the statistical power of such
studies and enhance the certitude of the
interpretations. Such an approach, in
combination with the technologies
mentioned above, should allow, in the
future, the expansion of a more comprehensive genetic testing strategy of
patients with the HPE phenotypic
spectrum and their relatives.
99
Finally, all of these considerations
contribute to difficulties in counseling
families with HPE [see Mercier et al.,
2010]. The extreme heterogeneity and
diverse manifestations of HPE presents
considerable challenges to medical
geneticists and counselors. No single
algorithm is presently sufficient to
explain all cases of HPE. However, we
hope that by providing a guideline for
the busy clinician, we can inspire the
clinical genetics community to engage in
fostering important research in this area.
The sharing of cases and case materials
should maximize the ability of clinicians
to provide meaningful results to their
patients for the present, as new technologies offer the future promise of an
even greater understanding of this complex set of malformations.
SUMMARY
In summary, our current recommendations for medically indicated genetic
testing of families with HPE is a tiered
approach with cytogenetic studies as the
first layer of the algorithm (see review by
Bendavid et al., this issue), since cytogenetic abnormalities make up the most
common causes of HPE. Molecular
testing of SHH, SIX3, and ZIC2 is
the second layer of evaluation, since they
explain at least 20% of non-syndromic
and non-chromosomal HPE. Other
genes identified in HPE should be tested
as complementary studies in special cases,
given their low frequency (1% or less).
These steps should take place in the
context of a discussion about whether to
pursue commercial lab testing and/or
enrollment in a research study.
ACKNOWLEDGMENTS
The authors thank the patients, families,
and clinicians from around the world for
their continued support of research
investigations into the genetic basis of
HPE and its clinical manifestations.
Likewise, we would like to acknowledge
Emily Kauvar (a Howard Hughes
Medical Institute (HHMI) scholar) for
critically reviewing this manuscript.
This work was supported by the Division of Intramural Research of the
National Human Genome Research
100
AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS)
Institute, the National Institutes of
Health.
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